Diversity of Rhizophydiales (Chytridiomycota) in Thailand: unveiling the hidden gems of the Kingdom

Chytrids, often overshadowed by their other fungal counterparts, take center stage as we unravel the mysteries surrounding new species within Rhizophydiales and explore their unique characteristics. In the broader spectrum of chytrids, their significance lies not only in their roles as decomposers but also as key players in nutrient cycling within aquatic ecosystems as parasites and saprobes. Baited soil and aquatic samples collected from various provinces of Thailand, yielded new species of the Rhizophydiales (Chytridiomycota), some of which expanded previously single species genera. Our investigation incorporated a combination of morphological and phylogenetic approaches, enabling us to identify these isolates as distinct taxa. The novel isolates possess distinguishing features, such as variations in size and shape of the sporangium and zoospores, that somewhat differentiate them from described taxa. To confirm the novelty of the species, we employed robust phylogenetic analyses using maximum likelihood and bayesian methods. The results provided strong support for the presence of eight distinct lineages within the Rhizophydiales, representing our newly discovered species. Furthermore, we employed Poisson Tree Processes to infer putative species boundaries and supplement evidence for the establishment of our new Rhizophydiales species. By meticulously exploring their morphological characteristics and genetic makeup, we expand the known catalogue of fungal diversity by describing Alphamyces thailandicus, Angulomyces ubonensis, Gorgonomyces aquaticus, G. chiangraiensis, G. limnicus, Pateramyces pingflumenensis, Terramyces aquatica, and T. flumenensis and also provide valuable insights into the intricacies of this order. This newfound knowledge not only enriches our understanding of Rhizophydiales but also contributes significantly to the broader field of mycology, addressing a critical gap in the documentation of fungal species. The identification and characterization of these eight novel species mark a noteworthy stride towards a more comprehensive comprehension of fungal ecosystems and their vital role.


Introduction
The kingdom Fungi comprises various distinct lineages namely, Dikarya, zygosporic and zoosporic taxa (Voigt et al. 2021;Wijayawardene et al. 2022).Among all the known fungal groups, zoosporic lineages are least studied (Hurdeal et al. 2020).Of these, the Chytridiomycota, commonly referred to as chytrids, is the most abundant and studied lineage (Letcher et al. 2015;Seto and Degawa 2018a;Longcore and Simmons 2020;Hurdeal et al. 2020;Voigt et al. 2021).Chytrids are predominantly found in aquatic systems.With the emergence of next generation sequencing data, we have evidence that chytrids represent a significant portion of the overall fungal community, however they are also isolated from terrestrial habitats (Panzer et al. 2015;Hassett et al. 2020;Longcore and Simmons 2020;Blaalid and Khomich 2021;Van den Wyngaert et al. 2022).Saprobic chytrids are detected on baits such as pollen grains, onion skin, and chitin among others (Longcore and Simmons 2020).Parasitic chytrids infect hosts ranging from green algae to amphibians in various aquatic niches (Fisher and Garner 2020;Seto et al. 2020a).
Identification of chytrids by light microscopy is often difficult because many species have similar morphology; consequently, species descriptions are primarily based on molecular phylogenetic analyses using the internal transcribed spacer and large subunit ribosomal ribonucleic acid (ITS-LSU) genetic markers (James et al. 2000) and previously ultrastructural details of the zoospores (Letcher et al. 2006).Recently, Hurdeal et al. (2023) proposed a polyphasic approach employing phylogeny, Poisson tree processes, and genetic distance analysis of the ITS to describe novel species (Schoch et al. 2012).This approach was proposed as morphology alone is insufficient to identify new taxa at the species level.
The systematics of Rhizophydiales has been continuously updated over the last few years.Molecular phylogeny and zoospore ultrastructure studies have enabled the elevation of the problematic genus Rhizophydium sensu lato to the order Rhizophydiales, within which Letcher, Powell and others, established new genera and neo-typed historical species (Letcher et al. 2006(Letcher et al. , 2008(Letcher et al. , 2012)).The historical chytrid literature has numerous morphologically described Rhizophydium species that are currently taxonomically unresolved because attempts to match new isolates to descriptions based on organisms not in axenic culture can be erroneous and therefore is problematic (Hurdeal et al. 2023).Intricacies, such as various species with different lifestyles, and the number of historical species makes this order particularly interesting to study.
Thailand is a tropical country with an immense diversity of flora, fauna, and funga with a significant potential for organismal discovery.In the last decade, descriptions of new fungal species from Thailand have contributed significantly to fungal taxonomy.Though most studies were and are still, primarily focused on dikaryan fungi, attempts to study basal lineages in the country are emerging (Hurdeal et al. 2021a(Hurdeal et al. , b, 2022(Hurdeal et al. , 2023)).To discover and explore zoosporic fungi, we collected and baited soil and water samples from different locations.We implemented a polyphasic approach using phylogeny, Poisson tree processes (PTP) and morphology to classify recovered Rhizophydiales isolates resulting in new species in Alphamyces, Angulomyces, Gorgonomyces, Pateramyces, and Terramyces.The new species and their phylogenetic relationships increase our knowledge of the Rhizophydiales in Thailand and also reveal the relationship of the new species to rhizophydialean species from other parts of the world.

Sample collection, and isolation
We collected soil and water samples from Chiang Rai, Chiang Mai and Ubon Ratchathani, Thailand.Water samples, along with some sediments, were collected from various lakes and rivers in sterile 50 mL centrifuge tubes.Soil samples were collected from the rhizosphere of shrubs and trees.Surface organic debris was manually removed and a sterile shovel or spoon was used to dig the soil, which was stored in Ziploc bags or centrifuge tubes.
For isolation, all samples were baited with pine pollen.Water samples were poured into 90 mm plastic Petri dishes, and pollen added. 1 g of soil was weighed and transferred to a sterile 90 mm Petri plate.The soil was then flooded with sterilized water and pollen added.All inoculated plates were kept at 20 °C and monitored daily under 100-400X magnification (Nikon Eclipse Ni).Once infected pollen grains were observed, a single sporangium was transferred with a drawn micropipette or a needle to PmTG agar medium supplemented with streptomycin sulfate (350 mg/L) and penicillin G (200 mg/L) (Barr 1986).Morphological characters of the fungi were observed with a Nikon Eclipse Ni compound microscope (100-600X) under DIC and images taken with a Nikon DS-RI2 digital camera.Isolates were preserved in 15% glycerol and following the protocol recommended by Collection of Zoosporic Eufungi at the University of Michigan (CZEUM) for long-term preservation (https:// czeum.herb.lsa.umich.edu/).Ex-type living cultures have been deposited in the Mae Fah Luang culture collection (MFLUCC), Chiang Rai, Thailand.Photoplates and species descriptions have been deposited in MycoBank.

DNA extraction, genetic marker amplification, and sequencing
Genomic DNA was extracted from mature cultures using G-spin ™ Total DNA Extraction Kit (Intron Biotechnology, South Korea) following the manufacturer's instructions.Amplicons of partial fragments of ITS1-5.8S-ITS2, and LSU were generated with polymerase chain reaction (PCR).The primers used were ITS4/ITS5 and LROR/ LR5 (Vilgalys and Hester 1990;White et al. 1990).PCR conditions were initial denaturation at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C of 1 min, annealing at 52 °C for 45 s, elongation at 72 °C for 90 s and final elongation at 72 °C for 7 min.
PCR products were purified with the MEGAquick spin plus fragment DNA purification kit (Intron Biotechnology, South Korea).Sequencing was performed with an Applied Biosystems 3130XL DNA analyzer (Bionics, South Korea).

Phylogenetic analysis and poisson tree processes
Raw DNA sequence data were edited and assembled into contigs using SeqMan Version 7.1.0.The newly generated sequences were used as queries to perform blast searches against the nucleotide database (nr) in GenBank to check for possible contamination and to assist with taxon sampling (Altschul et al. 1990).The dataset for Rhizophydiales followed Hurdeal et al. (2023) and was updated to include newly introduced taxa (see Table 1).Taxon sampling within identified genera where new species are being introduced spanned the genetic diversity currently available.Only ITS and LSU were used as genetic markers, as they are the most broadly available.Although, the small subunit ribosomal RNA (SSU) marker is also quite well represented for chytrids, for this order, the number of SSU sequence data available is low (< 40% of overall taxa used in this dataset).Hence the marker was not included in our analysis.Data for Rhizophydiales taxa were extracted from GenBank and CZEUM.Datasets for each genetic marker were built and aligned using MAFFT on the online webserver (https:// mafft.cbrc.jp/ align ment/ server/) and trimmed with TrimAl Version 1.2 (Katoh and Toh 2008;Capella-Gutiérrez et al. 2012).The two individual datasets were concatenated into a single matrix, which was used for the final phylogenetic analysis.
The IQ-TREE was computed on the webserver https:// iqtree.cibiv.univie.ac.at/ using the default parameters (Nguyen et al. 2015).Branch support was estimated from 1000 ultrafast bootstrap replicates.The analysis evaluated the best substitution model using Model Finder, which is embedded automatically in the analysis.The best model for Rhizophydiales was GTR + I + Γ for ITS and TIM3 + I + Γ for LSU.Maximum likelihood (ML) phylogeny using RAxML-NG Version 1.0.1 was inferred on the online CIPRES Portal with bootstrap support from 1000 pseudoreplicates (Miller et al. 2010;Kozlov et al. 2019).The combined data ML analysis was performed by partitioning the matrix according to the genetic markers (ITS and LSU) used and the best-suited models for each marker.The nucleotide substitution model for each genetic marker was evaluated using jModelTest2 on XSEDE in the online CIPRES Portal (https:// www.phylo.org/ porta l2) (Miller et al. 2010;Darriba et al. 2012).The best model under the AIC criterion was TPM2uf + I + Γ for ITS and GTR + I + Γ for LSU.Maximum likelihood phylogenetic analysis was performed for each dataset separately and the concatenated matrix.Bayesian inference (BI) analysis was performed using MrBayes Version 3.2.7a(Huelsenbeck and Ronquist 2001).Four simultaneous chains were run for 2 000 000 generations with a sampling frequency of 100.25% of the trees were discarded as burn-in.Convergence was declared when the standard deviation of split frequencies was less than 0.01.The final concatenated matrix and ML tree was deposited to Figshare (https:// doi.org/ 10. 6084/ m9.figsh are.24910 779).Newly generated sequences were deposited into GenBank (www.ncbi.nlm.nih.gov/ genba nk/).
To infer species boundaries, the coalescent-based Poisson tree processes (PTP) model was used (Zhang et al. 2013).PTP uses branch lengths (number of substitutions), which was extracted from the phylogenetic tree and added to infer branching events.The model assumes that the number of substitutions between species is significantly higher than within a species (Zhang et al. 2013).The analysis was performed on the online platform https:// speci es.h-its.org/ ptp/ and consisted of 100 000 Markov chain Monte Carlo (MCMC) generations, a thinning set to 100 and burn-in at 10%.The dataset comprised two genetic markers, ITS and LSU.Genus level ML (IQ-TREE) phylogenetic trees were computed.All analyses contained an outgroup, but a command to automatically remove distantly related outgroups to improve the delimitation results was implemented.Genetic distances (pairwise nucleotide substitution) were measured using the Kimura 2-parameter substitution model as implemented in MEGA-X with gamma distribution and pairwise deletion options.For the calculation, the trimmed alignments of ITS were used.

Results
We found chytrids on all of our pollen-baited samples.The overall morphology of the isolates was reminiscent to the members of the Rhizophydiales but we noticed slight differences in the culture morphology.Chytrids from the gross cultures grew readily on PmTG and mPmTG agar plates resulting in 11 isolates,  which represent eight new species from both terrestrial and aquatic habitats (Table 1).We constructed a Rhizophydiales phylogenetic tree (Fig. 1), with sequence data of 123 taxa from GenBank.In the final trimmed alignment, ITS comprised 713 and LSU 932 sites and the likelihood of the best scoring tree of the ML analysis was -31204.907.The topology of the phylogenetic trees from the RAxML, Bayesian, and IQ-TREE analyses was congruent.The placement of the novel taxa remained unchanged in  both single and concatenated matrix phylogenetic analyses.In all phylogenetic trees, the new strains formed clades distinct from the reference specimens.The position of nearly all new taxa was stable with high or maximum bootstrap support and posterior probability.
The PTP analysis results (Fig. 2) agreed with those of the inferred phylogeny concerning the novelty of the strains and species delimitation.Specifically, the PTP analysis indicated three distinct species within Angulomyces, eleven species in Gorgonomyces and six species in Terramyces.The genetic distances of the trimmed dataset of the new taxa and sister taxa were measured (Tables 2, 3, 4, 5 and 6).Angulomyces was represented by three clades and the genetic distance between them was 2-7% (Table 3).Gorgonomyces isolates grouped into eleven clades with genetic distance between clades ranging from 1.5 to 10.5% (Table 5).Following the phylogenetic species concept, Terramyces was split into four clades each representing a species; the average genetic distance between species was 1.1-9.2%(Table 6).In both Alphamyces and Pateramyces, PTP results indicate two clades with a interspecies genetic distances of 20% (Fig. 2; Tables 2 and 4).

Pateramyces pingflumenensis 20
Table 5 Genetic distance (%) between Gorgonomyces strains (grouped according to PTP results) in the ITS genetic marker (472 bp) Generic description: Sporangium spherical with a single discharge pore, the upper two thirds of the sporangial wall covered with long slender branched or unbranched hairs, sometimes slightly angular near the discharge papillae.Rhizoids branched.Zoospore contains a single, rather small lipid globule partially covered with a fenestrated cisterna.Mitochondrion single, a portion of which lies above and proximal to the kinetosome.Moderately electrondense walled vesicles occur in the cytoplasm adjacent to the kinetosome.Based on Letcher et al. (2008).
MycoBank: MB 848670 Etymology: Epithet references the country from where the species was isolated.
Notes: Phylogeny indicates clear distinction of the new isolate from A. chaetiferum with high statistical support obtained from maximum likelihood (IQ-TREE, RAxML) and Bayesian inference.The genetic distance between the types of A. chaetiferum and A. thailandicus in the trimmed ITS region is 20%.Distribution: Thailand.Angulomyces Letcher, Mycol. Res. 112(7): 776 (2008).MycoBank: MB 511779.Generic description and notes: See Hurdeal et al. (2023) and Letcher et al. (2008).
MycoBank: MB 848669 Etymology: Epithet refers to the province from where the species was isolated.
Notes: Phylogenetic analyses indicate three distinct species with maximum bootstrap support, and PTP specification.The pairwise nucleotide differences in the trimmed ITS (634 bp) of A. ubonensis to A. argentinensis and A. solicola are 7% and 6.5% respectively.
Distribution: Thailand.Gorgonomyces Letcher,Mycol. Res. 112 (7): 767 ( 2008)   Notes: Phylogenetic analyses and genetic distances show that G. aquaticus clusters with another unclassified strain of Gorgonomyces, hence providing more resolution to the delineation of the new species and insights on the distribution of this species.The genetic distance of this new species to other described taxa ranges from 4.8-5.5%.
Distribution: Thailand, and USA.
Diagnosis: Gorgonomyces limnicus is characterized by having smaller zoosporangia than G. haynaldii (ARG 026 -epitype) (to 50 µm in G. haynaldii), significantly fewer, shorter, and smaller discharge papillae but larger zoospores.This species differs from other Gorgonomyces strains introduced in this study and G. thailandicus, by having a different generation time, and larger sporangia.Notes: Phylogenetic analyses and genetic distances show that Gorgonomyces aquaticus is a new species.The distinct clading of the species to the other members is representative of a new species with a significant percentage pairwise difference in the ITS.The genetic distance of this new species to other described taxa ranges from 4.9-5.7%.MycoBank: MB 848674 Etymology: Epithet references the province from where the species was isolated.
Diagnosis: Gorgonomyces chiangraiensis has smaller sporangia and discharge papillae than G. haynaldii (ARG 026 -epitype) (to 50 µm in G. haynaldii).Gorgonomyces chiangraiensis produces more discharge papillae than G. thailandicus (to 4 in G. thailandicus), G. limnicus and G. aquaticus.This new species differs from other Gorgonomyces strains introduced in this study by having a different generation time, and larger sporangia.
Type: Thailand: Chiang Rai Province: Mae Chan District, from water baited with pine pollen, Jan. 2022, V.G.Notes: Phylogenetic analyses and genetic distances also indicate the novelty of G. chiangraiensis.The genetic distance of this new species to other described taxa ranges from 4.8-6.4%.
Notes: Pateramyces was introduced to accommodate three chytrids isolated from a water sample collected from a small lake in Argentina, and baited with pollen.Letcher et al. (2008), based the new genus on morpho-phylogenetic analyses.In the inferred ITS-LSU phylogenetic analysis, the taxon clustered sister to Rhizophydiaceae, leading to the introduction of a new family and genus.Morphological characterization indicated that Pateramyces corrientinensis isolates produced spherical sporangia at maturity, each with an operculate discharge tube.Pateramyces pingflumenensis possesses similar morphological characteristics.Genetic distance analyses (Table 4) in ITS further validated the introduction of the new species.
MycoBank: MB 848668 Etymology: The species epithet refers to the Ping River (Latin flumen = river), from which the strain was isolated.
Notes: The phylogeny indicates a clear distinction from P. corrientinensis with maximum statistical support obtained from maximum likelihood (IQ-TREE, RAxML) and Bayesian inference.The genetic distance between the type of P. corrientinensis and P. pingflumenensis in the trimmed ITS region is 20%.
Notes: Genetic analysis shows that this new species is clearly different from the type species in the genus.The genetic difference between this new species and others in the same genus is between 4.4% and 9.2%.PTP analysis confirms that this is indeed a new and distinct species.
MycoBank: MB 848676 Etymology: Epithet refers to the environment from where the species was isolated.
Diagnosis: This newly identified species differs from T. subangulosum (specifically the ARG-033 -epitype) by having larger sporangia (to 67 µm diam.), however, it has smaller sporangia and zoospores compared to T. flumenensis.
Notes: Genetic analyses reveal that this new species forms a separate branch distinct from the type species and other described species in the genus.The genetic distance in the trimmed ITS between the novel species and others in the same genus ranges from 4.4% to 7.7%.Additionally, PTP analysis confirms the uniqueness of this newly discovered species.
Distribution: Thailand, and USA.

Discussion
Chytrid taxonomy has experienced significant changes over the years.Early taxonomy was based on the morphological species concept (e.g., Sparrow 1943Sparrow , 1960;;Karling 1977).In the 1980s, the features used for the identification of chytrids changed drastically with the implementation of zoospore ultrastructure based on transmission electron microscopy (Barr 1980).Currently, the gold standard for the establishment of new taxa incorporates both morphological and molecular data.Species delimitation can bypass ultrastructure data because resolution provided by TEM is not definitive at the species level (Hurdeal et al. 2023).Furthermore, obtaining good quality ultrastructure data is often a bottleneck as not all species produce zoospores abundantly.Also, the expertise of interpreting zoospore ultrastructure data is limited, and the equipment not widely accessible to researchers.Hence, this slows down progress in describing the largely uncharacterized chytrid diversity (Tables 5 and 6).
Diversity and distribution information for chytrids is significantly lower relative to that for members of the Dikarya.However, steady progress has been made in the last twenty years, with many studies from the Americas (Letcher et al. 2008;Longcore 2004Longcore , 2011;;Simmons et al. 2009Simmons et al. , 2020Simmons et al. , 2021;;Wakefield et al. 2010;Marano et al. 2011;Longcore and Simmons 2012;Longcore et al. 2012;Vélez et al. 2013;Davis et al. 2015).Descriptions of new taxa are also coming from other parts of the globe (Seto and Degawa 2018a, b;van den Wyngaert et al. 2018;Hyde et al. 2019;Seto et al. 2020a, b;Karpov et al. 2021;Hurdeal et al. 2023).Collectively, these studies depict a broad distribution of chytrids in various parts of the world.
In this study, we increase the global knowledge of chytrids by introducing eight new rhizophydialean species from Thailand.Delineation of these species is based on a tripartite approach including morphological characterization, phylogenetic analyses based on ITS-LSU genetic markers and PTP.The morphology of the new species differs somewhat from the described species within the genera.Differences include the sizes of the reproductive structures and other morphological characters such as number of discharge papillae.However, because morphological characters are few and differ only slightly, we place the most emphasis on phylogenetic analyses of molecular data.
Phylogeny provided high statistical support for the establishment of most of the new taxa except in Terramyces, for which statistical support is low.This may be indicative of low resolution of the ingroups, or problematic sequences.Hence, in total in this study, the PTP analysis divided the currently known Terramyces strains into six groups, each group representative of a separate species.This reflects the effects of taxon sampling and the relative genetic distance among and within species.In all analyses, however, the placement of strains and taxa was stable.In Terramyces, the observed pairwise nucleotide differences spanned from 1.1% to 9%.The 1.1% divergence appears relatively low for the delineation of a new species.PTP suggests the possibility that groups 1-3 may indeed constitute a single taxonomic entity.This underscores the importance of employing the PTP method repeatedly as new strains and species are uncovered.Hence, the iterative application of PTP Similarly, in Gorgonomyces the type strain Gorgonomyces haynaldii ARG 026 segregated from ARG 024, whereas previously the two grouped together.Hence, as Hurdeal et al. (2023) suggested, phylogenetic analysis is a dynamic process and as new strains become available, analyses are needed to validate the use of PTP or any new phylogenetic tools.Our results indicate that not only is generic diversity high but diversity is also high at the species level.
For the rest of the concerned monospecific genera, the PTP provided indication and evidence for the novelty of our isolates.However, as more strains and species become available, the analyses may need to be re-evaluated.Our new Pateramyces isolate differs by 20% from the P. corrientinensis clade in the trimmed ITS genetic marker indicating a high degree of genetic diversity in the genus.
Pateramyces and Alphamyces were previously monotypic genera isolated from Argentina.Letcher et al. (2008), introduced family and genus based on three isolates.From currently known data, the two genera are saprobes on pollen grains, whereas their diversity and distribution seem to be restricted to aquatic environments.Hence, it is evident from this and previous studies, that our knowledge of chytrid diversity remains quite limited.Consequently, the precision of the methods employed and their morphological characteristics are still uncertain.Future research will require thorough investigation into the ecology, re-evaluation of various taxonomic ranks, and the study of historical morphological species in order to gain a comprehensive understanding of this group of fungi.

Table 1
Data used for phylogenetic analysis of Rhizophydiales in this study, their corresponding GenBank accession numbers, source and habitats.Type (T), epitype (ET), ex-type (EX), and neotype (NT) species are denoted by superscripts to species names.Sequences derived in this study are shown in bold

Table 2
Genetic distance (%) between Alphamyces strains (grouped according to PTP results) in the ITS genetic marker (603 bp)

Table 6
Genetic distance (%) between Terramyces strains (grouped according to PTP results) in the ITS genetic marker (568 bp)